The long-term objectives of this project are to determine the mechanisms by which interleukin 3 (IL-3) regulates the growth and differentiation of hematopoietic cells. The research focuses on the initial biochemical events that occur following IL-3 binding to its receptor and, based on studies demonstrating the essential role of tyrosine phosphorylation, seek to identify critical substrates of these reactions. Studies during the previous granting period identified a member of the Janus family of cytoplasmic tyrosine kinases, Jak2, as associating with the IL-3 receptor and being activated by IL-3. Studies during the last year demonstrated that the deletion of Jak2 in mice eliminates the response to IL-3. Additional studies indicated that critical biochemical events in IL-3 signaling were dependent upon the recruitment of substrates to the receptor complex through interactions with sites of tyrosine phosphorylation on Jak2. Therefore, during the last year the major sites of tyrosine auto- phosphorylation on Jak2 have been identified. In the proposed studies, we seek to identify the proteins that are recruited to the receptor complex by interaction with sites of tyrosine phosphorylation on Jak2. To accomplish this we propose to use a variety of approaches that have proven to be successful in both my laboratory and in the laboratories of investigators studying other receptor systems. The approaches utilized will include yeast two-hybrid screening, the screening of libraries with phosphopeptides or proteins or the use of affinity columns. From these approaches we have already identified a number of potentially interesting Jak2 associating proteins including JAB1, JAB2, p110delta, PLC-gamma2 and a novel protein. Based on these results we anticipate that a number of additional proteins will be identified. In the second specific aim of this application we propose experiments that seek to determine the significance of the association of these proteins with the IL-3 receptor complex and Jak2. A variety of standard approaches will be used to look at expression patterns, chromosomal localization, cellular localization and approaches to confirm the associations in cells in response to IL-3. Central to our studies however will be the derivation of mice in which the individual genes have been disrupted by homologous recombination in embryonic stem cells. Such mutant mouse strains have already been derived for JAB1, JAB2 and PLC-gamma2 and are being characterized. Together the studies will continue to address fundamental questions regarding the biochemical mechanism by which IL-3 regulates the growth and differentiation of hematopoietic cells as well as potentially providing insights into signal transduction by a wide spectrum of cytokines that utilize receptors of the cytokine receptor superfamily.